USPTO Patents Application 10687850

J

Europaisches Patentamt
European Patent Office
Office europeen des brevets

@ Publication number:

0 255 291 B1

©

EUROPEAN PATENT SPECIFICATION

© Date of publication of patent specification: 24.06.92 © Int. CI. 5 G01N 27/42, C12M 1/40
© Application number: 87306513.0
@ Date of filing: 23.07.87

Divisional application 91117022.3 filed on
23/07/87.

© Method and apparatus for electrochemical measurements.

© Priority: 23.07.86 GB 8618022

@ Proprietor: UNILEVER PLC

Unilever House Blackfriars P.O. Box 68

@ Date of publication of application:

London EC4P 4BQ(GB)

03.02.88 Bulletin 88/05

© Designated Contracting States:

GB

© Publication of the grant of the patent:

24.06.92 Bulletin 92/26

© Proprietor: UNILEVER NV

© Designated Contracting States:

Burgemeester sMacobplein 1 P.O. Box 760

NL-3000 DK Rotterdam(NL)

AT BE CH DE ES FR GB GR IT LI NL SE

© Designated Contracting States:

BE CH DE ES FR GR IT LI NL SE AT

© References cited:

EP-A- 0 078 636

© Inventor: Birch, Brian Jeffrey

EP-A- 0 125 137

14 Duchy Close

EP-A- 0 177 743

Chelveston Northamptonshire, NN9

DE-B- 1 932 581

6AW(GB)

Inventor: Burns, Ian William

AGRICULTURAL AND BIOLOGICAL CHEMIS-

The Little House Shelton

TRY, vol. 48, no. 8, August 1984, pages

Huntingdon Cambridgeshire PE18 ONP(GB)

1969-1976, Tokyo, JP; T. IKEDA et aL:

"Electrocata lysis with a gluose-

oxidase-immobtlized graphite electrode"

© Representative: Butler, David John et al

Unilever PLC Patents Division P.O. Box 68

Unilever House

London EC4P 4BO(GB)

cn
cm

in
in
cm

a.
ui

Note: Within nine months from the publication of the mention of the grant of the European patent, any person
may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition
shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee
has been paid (Art. 99(1) European patent convention).

Rank Xerox (UK) Business Services

EP 0 255 291 B1

ANALYTICAL CHEMISTRY, vol. 56, no. 2, Feb-
ruary 1984, pages 148-152, Easton, Pennsyl-
vania, US; H. DURLIAT et al.: "Amperometrlc
enzyme electrode for determination of glu-
cose based on thin-layer spectroelec-
trochemlstry of glucose oxidase**

ANALYTICAL CHEMISTRY, vol. 50, no. 7, June
1978, pages 944-950, Easton, Pennsylvania,
US; R.E. ADAMS et al.: "Coulometrlc flow
analyzer for use with Immobilized enzyme
reactors"

INDUSTRIAL LABORATORY, vol. 46, no. 2,
February 1980, pages 116-118, New York, US;
T.K. KHAMRAKULOV et al.: "Coulometrlc de-
termination of chlorine"

JOURNAL OF PHYSICS E. SCIENTIFIC IN-
STRUMENTS, vol. 18, no. 9, September 1985,
pages 736-749, Bristol, GB; C. NYLANDER
"Chemical and biological sensors"

1

EP 0 255 291 B1

2

Description

Field of invention

This invention relates to methods and appara-
tus for making electrochemical measurements, in
particular but not exclusively for the purpose of
carrying out microchemical testing on small liquid
samples of biological, e.g. clinical origin.

Background to the Invention

F. Schlapfer et al (Clin.Chim.Acta, 1974, pp
283-289) described electrochemical measurement
of glucose concentration using glucose oxidase
and soluble electron transfer substances such as
ferricyanide, p-benzoquinone, 2,6-dichlorophenolin-
dophenol, pyocyanine, thionine or methylene blue,
interacting with amperometric noble-metal elec-
trodes.

These arrangements have not given rise to
glucose-measurement products which are simple
and convenient to use in environments far removed
from the skilled inhabitants of analytical laborato-
ries.

Since 1974 a variety of further electrode ar-
rangements have been proposed for
chemical/immunochemical analysis, among them
electrodes carrying immobilised redox mediators
as well as enzymes, for example EP 0 078 636, 0
125 136 and 0 125 139 (Genetics International),
and 0 142 301 (Serono). EP 0 177 743 (Shimadzu)
describes enzyme electrodes of somewhat com-
plex construction, which are capable of use to
measure a number of enzyme substrates by am-
perometry, using electron transfer mediators.

EP 0 125 137 discloses an electrode sensing
system, for monitoring components in a liquid mix-
ture, using a probe-type sensor which typically
comprises an electrode surface carrying a gen-
erally insoluble electron transfer mediator, in turn
coated with an enzyme, the electron transfer me-
diator acting to transfer charge between the en-
zyme and the electrode. The system is typically
used to determine glucose concentrations, using
amperometric techniques.

The paper in "Analytical Chemistry \ Volume
56, No 2, February 1984, pages 148 to 152 dis-
closes an enzyme electrode using a thin layer
platinum electrode in conjunction with the enzyme
glucose oxidase or flavine adenine dinucleotide,
and refers to the possibility of reducing the depth
of the reaction chamber to optimize performance.

This invention aims to provide measurement
apparatus and methods to enable quick, convenient
and accurate measurement of various constituents
of liquid samples, especially of biological origin,
e.g. clinical samples of blood, serum, or urine.

The invention also aims to provide measure-
ment apparatus of simple construction which can
be treated as disposables.

It is also an aim of the invention to enable
5 convenient and in many cases quantitative elec-
trochemical methods to be applied to analytes
which do not themselves react with electrodes.

It is a further aim of the present invention to
provide electrochemical apparatus for convenient
io liquid sample analysis but without requiring com-
plex electrode structures involving immobilised
components.

Summary of the invention

15

According to one aspect of the invention there
is provided a method for measurement or detection
of a component of an aqueous liquid sample, said
method comprising:

20 placing a sample, possibly containing the compo-
nent of interest, into a capillary fill reaction cell,
said reaction cell comprising at least two elec-
trodes adapted to coulometric measurements, so
that the sample forms a layer of liquid having a

25 thickness less than about 0.2 millimeter in a reac-
tion zone overlying one of said electrodes,
reacting said component, if present, directly or
indirectly with a redox reagent initially present in
the form of a solid releasable layer deposited on an

30 internal surface of the reaction cell and which is
released into solution in said sample in said zone,
thereby to form in said zone in a quantity propor-
tional to the amount of said component present in
said zone of an electrochemically oxidizable or

35 reducible substance capable of reacting electro-
chemically with said electrode, and being different
from said redox reagent, and
coulometrically detecting or estimating a quantity
of electrical charge which relates to said oxidizable

40 or reducible substance in said zone to provide an
indication of the amount of said component of
interest.

The component to be analysed may oxidise or
reduce directly or indirectly an electron transfer
45 substance which is then estimated electrochemi-
cally. The production of the electrochemically ox-
idisable or reducible substance may be made to
occur enzymatically.

The result relating to the quantity of the sub-
50 stance to be measured can be of use, among other
things, as an index of the concentration of the
substance in a liquid sample.

In this specification and claims 'redox reagent'
and similar terms and 'electron transfer reagent*
55 and corresponding terms are mutually inclusive.

Also provided by the invention is apparatus for
carrying out electrochemical detection or measure-
ment of a component of an aqueous liquid sample,

3

3

EP0 255 291 B1

4

said apparatus comprising:
a capillary fill reaction cell comprising at least two
electrodes, the electrodes being adapted to
coulometric measurements and being located in or
adjacent to a reaction zone, said reaction zone 5
being capable of receiving an aqueous liquid sam-
ple possibly containing said component of interest,
and said electrodes and said reaction zone being
arranged so that liquid in said zone contacts said
electrodes and forms a layer having a thickness ro
less than about 0.2 millimeter overlying one of said
electrodes,

and said cell also comprising a redox reagent in
the form of a solid releasable layer deposited on an
internal surface of the cell and located to contact 75
said sample when said sample is introduced into
said cell and into said reaction zone, said redox
agent being released into solution in said aqueous
sample in said zone and reacting directly or in-
directly with said component to form an electro- 20
chemically oxidizable or reducible substance in
said zone, said oxidizable or reducible substance
being capable of reacting electrochemically with
said electrode and being different from said redox
reagent. 25

It is especially preferred to provide in the use
of this method a cell which confines the liquid
reagents to react with the electrode to a sufficiently
thin layer overlying the electrode to permit
coulometric measurement of the electro-active ma- 30
terial to take place in a short time. A suitable
thickness for the liquid layer is for example of the
order of about 0.02 to 0.2 mm, for example about
0.1 mm. Capillary-fill cells with a configuration as
described in EP 0 170 375 (Unilever) are among 35
the cells suitable in this respect. In certain useful
arrangements within the scope of the invention, the
cell may confine a defined reactive volume of sam-
ple or reaction liquid in a space of defined width
between a cell wall and an electrode of defined aq
area. Liquid outside the volume may be able to
diffuse inwards but for example only at an inappre-
ciable rate compared to the time required for reac-
tion of the liquid in the defined volume. In other
useful embodiments, the cell may define a volume 45
of liquid to provide material to react at the elec-
trode.

In one preferred kind of test arrangement, the
component of the aqueous liquid sample to be
measured is a reducible sugar such as glucose, 50
and a reagent with which said sugar is allowed to
react is an oxidase enzyme specific for said sugar
desired to be measured, such as glucose oxidase,
together with any further substrate. Glucose and
glucose oxidase together react with an electron 55
transfer mediator such as ferricyanide ion in place
of their normal further substrate oxygen to produce
a corresponding quantity of ferrocyanide ion. Fi-

nally the ferrocyanide ion so produced is estimated
coulometrically by anodic oxidation.

Among further suitable examples of electron
transfer substances are methylene blue, p-ben-
zoquinone, 2,6-dichlorophenolinophenol,
pyocyanine and thionine.

The component to be measured can in general
be formed by initial enzymic or chemical conver-
sion of an analyte: e.g. an analyte can be sucrose,
and invertase can be contained in said cell to form
from said sucrose glucose by hydrolysis: the glu-
cose so formed can then be measured by the
methods described herein.

An oxidoreductase enzyme can be present in
said cell to mediate any desired reaction between
the component to be measured and any additional
electron transfer reagent.

In one class of tests which can be carried out
using the devices and methods described in more
detail herein, the component to be measured com-
prises an electrochemically reducible metal ion or
an electrochemically oxidisable inorganic ion or an
electrochemically oxidisable or reducible organic
compound and is measured either by direct
coulometry or by coulometry after initial electro-
chemical conversion to an electrochemically ox-
idisable or reducible intermediate. It may not in all
cases be necessary to deplete completely the
electroactive species to be measured.

The form of the reaction cell in which these
reactions are allowed to take place can contribute
significantly to the convenience of the test proce-
dure. It is preferred to use an adapted form of the
capillary fill cells provided with electrodes as de-
scribed in European Specification No 0 170 375
(Unilever), containing electrodes of suitable imped-
ance carried as thin films on one or more walls
thereof. The drawings and description of said
specification are incorporated herein by reference,
to be modified by the indications given herein for
making and using the measurement devices and
methods of the present invention.

Such a cell as adapted for the purposes of the
present invention can suitably for example com-
prise three electrodes, viz (a) a working electrode,
for example of gold or other noble metal, carbon or
graphite in any convenient form, e.g. wax-impreg-
nated graphite; (b) a counterelectrode, chosen from
a similar range of materials as given for electrode
(a), and possibly of the same materia! as electrode
(a) itself; and (c) a reference electrode, for examble
a silver/chloride electrode, or pH electrode.

Choice of electrode materials can for many
purposes preferably be made among gold, silver
and carbon film electrodes.

In one convenient form, both or all electrodes
are contained as films in a capillary fill cell formed
between two parallel flat plates spaced apart by

4

5

EP 0 255 291 B1

6

about 0.1 mm cell thickness, with about 0.1 mm
thick tracks of sealing material forming the remain-
ing sides of the cell apart from an aperture for
entry of liquids.

It can be convenient to form such a cell using
opposed plates of for example ceramic, plastics or
glass. When such a cell is fabricated, as is pre-
ferred, by the use of ceramics, a suitable substrate
can be for example a 96% alumina substrate
(Kyocera A4476 - Trade Mark), and a preferred
material for the electrodes to be formed thereon is
gold, applied as gold printing paste (Engelhard
T4474 - Trade Mark), to be applied in a high
temperature oxidative furnace in accordance with
the ordinary methods of use of that material. This
results in the context of this invention in a gold
layer with overlying thin oxide layer capable of
constituting a highly reproducible electrode. When-
ever desired, part of the metal layer can be
blanked off by overlying, dielectric layers e.g.
formed of dielectric printing ink (DuPont 5704 -
Trade Mark) applied to the substrate according to
the ordinary manner of use of that material.

When reactions of the kinds described above
are allowed to occur in a cell as described above, it
is found that a working electrode can easily deplete
substantially all of the electroactive material in that
part of the liquid that overlies the working elec-
trode, before any substantial lateral diffusion has
taken place.

Accordingly, it is preferred to use cells of such
dimensions that this situation prevails: i.e. that the
time required for lateral diffusion of an appreciable
amount of reactive material from the region outside
that which overlies the working electrode, to the
region overlying the working electrode, is much
longer than the time required for diffusion of cell
contents across the thickness of the cell and for
depletion by an electrode of the material capable of
reacting with it from the region of the cell overlying
said electrode.

An advantage arising from use of the invention
in this manner is that the measurement can be
made substantially insensitive to the nature of the
electrode material and calibration of the measure-
ments can be particularly simple and uniform as
between samples of the devices as described here-
in.

The arrangements of the invention can for ex-
ample take the form of coulometric measurements.
Such measurement methods are in themselves
known and their details do not constitute the
present invention.

Further details are given below in connection with
the following illustrative example.

Example

Examples of glucose measurement will now be
described non-limitatively, first in a coulometric
embodiment.

Reagents for the test can conveniently be dried
5 down on to a surface which either forms part, or
will form part, of a glass or ceramic inner surface of
a capillary fill cell.

The reagents can be dried down either by
filling reagent liquid into a pre-formed cell (e.g. 0.1

w mm wide) and then drying, or by screen-printing a
liquid layer up to 0.1 mm thick to be dried on to
said surface which will form part of said cell when
said cell is fabricated from a component carrying
dried printed reagents.

;s In the present example the reagents are cho-
sen so that upon rehydration in the sample liquid
filling the cell they give:-
buffer (preferably about 0.1 M ammonium citrate,
otherwise e.g. 0.5M sodium phosphate) adjusted to

20 approximtely neutral pH;

0.5M potassium ferricyanide; and

0.5 mg/ml glucose oxidase (a considerable excess,

which may be reduced).

Low molecular weight (about 40,000) polyvinyl-

25 pyrrolidone can be used as a carrier and/or
stabiliser, used in a quantity and concentration
dictated largely by the volume of reagent liquid to
be applied and dried, and by the method of ap-
plication, e.g. at 5% w/v in liquid to be filled and

30 dried in a preformed cell, and at higher concentra-
tion (optionally lower volume) in liquid to be print-
ed. In other variants, any other reagents can also
be present to suit the test to the test sample liquids
to be used - e.g. further anticoagulant besides

35 citrate, if necessary, where whole blood is to be
tested. This example gives a sensitivity range of
about 0 to 20 mmolar glucose concentration. In
other variant examples, suitable concentrations for
the ferricyanide lie in the order of about 3 times the

40 maximum concentration of glucose to be estimat-
ed. Chloride ion should be present where a chlo-
ride electrode is used as the reference electrode.
Also usefully present in certain variants can be an
inhibitor of catalase, e.g. sodium azide, and/or a

45 chemical deoxygenator.

The dried reagents may be carried and/or
stabilised on a surface by inclusion of a water-
soluble polymer, e.g. polyvinylpyrrolidone, or alter-
natively a water-insoluble polymer support such as

so a thin layer of cellulose acetate.

Test liquid is introduced into the cell. The
immobilised reagents including the electron trans-
fer substance (ferricyanide) are allowed to dissolve
and disperse throughout the volume of the test

55 liquid, and the reaction of the glucose and the
glucose oxidase is allowed to take place, reducing
the ferricyanide to ferrocyanide.

The electrical arrangements can comprise for

5

7

EP 0 255 291 B1

8

example a conventional potentiostatic control ar-
rangement in which, for example, a voltage-follower
impedance transformer is connected with its input
taken from the working electrode and reference
electrode, and its output taken, through circuitry to 5
apply a working low-impedance voltage between
the working electrode and the counterelectrode, in
a negative feedback arrangement such that the p.d.
between the working electrode and reference elec-
trode is kept close to a desired level. 10

A current integrator is connected with its inputs
taken from the working electrode and counterelec-
trode, and delivers as its output a signal which is to
be taken as the coulometric measurement given by
the device. 75

Initially, the potentiostatic control is set so that
the p.d. between working and reference electrode
is insufficient to allow electrode reaction of the form
of electron transfer substance produced indirectly
by reaction of the analyte: at a point in time from 20
which the coulometric integration is to be started,
the voltage is stepped to a level that does allow
such electrode reaction. Typically, a potential at the
working electrode is chosen that oversteps the
redox potential of the electron transfer material by 25
of the order of about 0.05 - 0.1 volt, to maximise
the wanted reaction relative to any side reactions.
Then the current is monitored and for example
integrated for a wanted appropriate interval of time
to provide the desired signal indicative of the want- 30
ed measurement. Suitable 'inactive' and 'active'
potentials for the ferro/ferricyanide embodiments
can be for example in the range up to about +
0.25 volt and + 0.5 volt respectively.

A preferred configuration for this and other 35
examples involves the use of a cell comprising a
pair of gold electrodes. In this case one gold elec-
trode can serve as a counter-electrode as well as a
reference electrode, and a substantially invariant
potential can be obtained via the ferricyamide aq
present in the reaction mixture, of which the quan-
tity can most suitably be large, (e.g. much larger
than the quantity of analyte and ferrocyamide
formed by reduction,) hence substantially constant.
A preferred operating potential can be at about + 45
0.15 volt.

It can be especially convenient to provide a
simple combination of potentiostat and digital me-
ter readout for the integrator. Then the user can
watch until the digital reading comes substantially 50
to a standstill (i.e. upon completion of the electrode
reaction) and takes the reading at that point as the
wanted measurement result, or in another arrange-
ment, involving automatic data processing, the digi-
tal signal can be stored when its rate of change 55
has subsided below a preset threshold rate.

It is found that typical electrode currents in this
coulometry are of the order of fractions of a mil-

liamp for a few minutes where typical blood glu-
cose concentrations are measured, e.g. in whole
blood, plasma or serum.

In one alternative variant of this embodiment,
there may be no 'inactive' potential applied to the
cell formed by the working and counter electrodes,
but rather this cell may be left open-circuit until the
current integration is to take place.

In a variant of the process mercuric ions can
be measured using a gold electrode by introducing
a sample into a capillary fill cell, and estimating a
trace quantity of mercury by step coulometry. This
comprises holding the electrode potential more
positive than the mercuric ion reduction potential,
e.g. 4 0.5 volt, until other current-generating pro-
cessess have decayed. Then the voltage is
stepped to a value more negative than the mercu-
ric ion reduction potential, e.g. to -0.1 volt. Then
the charge passed until the current decays can be
taken as an index of the mount of mercury present.

In further embodiments, it can be sufficient to
use a working electrode and a further electrode
combining the functions of reference and counter
electrode, provided that the further electrode, is a
metal/metal-halide reference electrode and the cor-
responding halide is present in the sample, prefer-
ably at standardised concentration. Alternatively,
any other further electrode of low electrochemical
impedance and adequately-defined potential may
be used. Suitable electrodes for use are for exam-
ple as described in EP 0 186 286 (Unilever).

The invention described herein is susceptible
of many modifications and variations as will be
apparent to the skilled reader, and the disclosure
herein extends to the use of all combinations and
subcombinations of the features as claimed.

Claims

1. A method for measurement or detection of a
component of an aqueous liquid sample, said
method comprising:

placing a sample, possibly containing the com-
ponent of interest, into a capillary fill reaction
cell, said reaction cell comprising at least two
electrodes adapted to coulometric measure-
ments, so that the sample forms a layer of
liquid having a thickness less than about 0.2
millimeter in a reaction zone overlying one of
said electrodes,

reacting said component, if present, directly or
indirectly with a redox reagent initially present
in the form of a solid releasable layer depos-
ited on an internal surface of the reaction cell
and which is released into solution in said
sample in said zone, thereby to form in said
zone a quantity proportional to the amount of
said component present in said zone of an

6

9

EP 0 255 291 B1

10

efectrochemically oxidizable or reducible sub-
stance capable of reacting electrochemically
with said electrode, and being different from
said redox reagent, and

coulometrically detecting or estimating a quan-
tity of electrical charge which relates to said
oxidizable or reducible substance in said zone
to provide an indication of the amount of said
component of interest.

2. A method according to claim 1, wherein said
component to be measured is formed by initial
enzymatic or chemical conversion of an an-
alyte.

3. A method according to claim 1 or 2, wherein
said redox reagent is selected from the group
comprising ferricyanide, methylene blue, p-be-
nzoquinone, 2,6-dichlorophenolindophenol,
pyocyanine and thionine, and said electro-
chemically oxidizable or reducible substance is
oxidizable and comprises a reduced form of
said redox reagent.

4. A method according to any one of claims 1 to
3, wherein said component is reacted with an
enzyme and said redox reagent thereby to
form said electrochemically oxidizable or re-
ducible substance.

5. A method according to claim 4, wherein said
enzyme comprises an oxidoreductase enzyme.

6. A method according to claim 4 or 5, wherein
said component comprises a reducing sugar
and said enzyme comprises a corresponding
sugar oxidase.

7. A method according to claim 6, wherein said
component comprises glucose and said en-
zyme comprises glucose oxidase.

a Apparatus for carrying out electrochemical de-
tection or measurement of a component of an
aqueous liquid sample, said apparatus com-
prising:

a capillary fill reaction cell comprising at least
two electrodes, the electrodes being adapted
to coulometric measurements and being lo-
cated in or adjacent to a reaction zone, said
reaction zone being capable of receiving an
aqueous liquid sample possibly containing said
component of interest, and said electrodes and
said reaction zone being arranged so that liq-
uid in said zone contacts said electrodes and
forms a layer having a thickness less than
about 0.2 millimeter overlying one of said elec-
trodes,

and said cell also comprising a redox reagent
in the form of a solid releasable layer depos-
ited on an internal surface of the cell and
located to contact said sample when said sam-

5 pie is introduced into said eel! and into said

reaction zone, said redox reagent being re-
leased into solution in said aqueous sample in
said zone and reacting directly or indirectly
with said component to form an electrochemi-

/o cally oxidizable or reducible substance in said

zone, said oxidizable or reducible substance
being capable of reacting electrochemically
with said electrode and being different from
said redox reagent.

75

9. Apparatus according to claim 8, further com-
prising an enzyme located to contact said
sample when said sample is introduced into
said cell and into said reaction zone, said

20 enzyme being capable of catalysing direct or

indirect reaction between said component and
said redox reagent.

10. Apparatus according to claim 9, wherein said
25 enzyme is carried as a releasable layer on a

surface of said reaction cell or reaction zone
which in use is contacted by aqueous sample
introduced into said apparatus.

30 11. Apparatus according to claim 9 or 10, wherein
said enzyme comprises an oxidoreductase.

12. Apparatus according to claim 11, wherein said
enzyme comprises glucose oxidase.

35

13. Apparatus according to any one of claims 8 to

12, wherein said redox reagent is selected
from the group comprising ferricyanide,
methylene blue, p-benzoquinone, 2,6-dich-

40 iorophenolindophenol, pyocyanine and

thionine, and said electrochemically oxidizable
or reducible substance is oxidizable and com-
prises a reduced form of said redox reagent.

45 14. Apparatus according to any one of claims 8 to

13, wherein said electrodes are selected from
the group comprising gold, carbon and silver
electrodes.

so Revendications

1. Procede de mesure ou de detection d'un com-
posant d'un echantillon liquide aqueux, ledit
procede consistant a:
55 placer un echantillon, contenant eventuel-

lement le composant interessant, dans une cel-
lule a reaction a remplissage capillaire, ladite
cellule a reaction comprenant au moins deux

7

11

EP 0 255 291 B1

12

Electrodes adapters aux mesures coulomEtri-
ques, afin que i'echantillon forme une couche
de liquide ayant une Epaisseur inferieure a
environ 0,2 millimetre dans une zone rEaction-
nelfe recouvrant une desdites Electrodes,

a faire rEagir ledit composant, si present,
directement ou indirectement avec un rEactif
redox initialement present sous la forme d'une
couche solide libErable dEposEe sur une surfa-
ce interne de la cellule rEactionnelle et qui est
libErE dans la solution dans ledit Echantillon
dans ladite zone, pour former de cette fagon
dans ladite zone une quantite* proportionnelle a
la quantity dudit composant present dans ladi-
te zone d'une substance oxydable ou rEducti-
ble Electrochimiquement capable- de rEagir
Electrochimiquement avec ladite electrode, et
Etant diffErente dudit rEactif redox, et

a dEtecter ou a estimer coulometrique-
ment une quantity de charge electrique qui est
fonction de ladite substance oxydable ou re-
ducible dans ladite zone pour fournir une indi-
cation de la quantite" dudit composant intEres-
sant

2. Procede selon la revendication 1, dans lequel
ledit composant a mesurer est forme" par la
conversion enzymatique ou chimique initiate
d'un analyte.

3. Procede selon la revendication 1 ou 2, dans
lequel ledit rEactif redox est choisi parmi ie
groupe constituE de ferricyanure, du bleu de
methylene, du p-benzoquinone, du 2,6-dichlo-
rophenolindophenol, de la pyocyanine et de la
thionine, et ladite substance oxydable ou rE-
ductible electrochimiquement est oxydable et
comprend une forme reduite dudit rEactif re-
dox.

4. ProcEdE selon Tune quelconque des revendi-
cations 1 a 3, dans lequel ledit composant est
mis a reagir avec une enzyme et ledit rEactif
redox pour former de cette fagon ladite subs-
tance oxydable ou reducible electrochimique-
ment.

5. Procede selon la revendication 4, dans lequel
ladite enzyme comprend une enzyme oxydore-
ductase.

6. Procede" selon la revendication 4 ou 5, dans
lequel ledit composant comprend un sucre rE-
ducteur et ladite enzyme comprend un sucre
oxydase correspondant.

7. Procede selon la revendication 6, dans lequel
ledit composant comprend Ie glucose et ladite

enzyme comprenant la glucose oxydase.

8. Appareil pour rEaliser la detection ou la mesu-
re Electrochimique d'un composant d'un

s Echantillon liquide aqueux, ledit appareil etant

constituE de:

une cellule a reaction a remplissage capil-
laire comprenant au moins deux Electrodes,
!es Electrodes Etant adaptEes aux mesures

io coulomEtriques et Etant situEes dans ou, adja-

centes a une zone rEactionnelle, ladite zone
rEactionnelle Etant capable de recevoir un
Echantillon liquide aqueux contenant Eventuel-
lement ledit composant intEressant, et lesdites

is Electrodes et ladite zone rEactionnelle etant

disposEes de fagon que Ie liquide dans ladite
zone soit en contact avec lesdites Electrodes
et forme une couche ayant une Epaisseur infe-
rieure a environ 0,2 millimetre s'Etendant sur

20 une desdites Electrodes,

et ladite cellule comprenant Egalement un
rEactif redox sous la forme d'une couche soli-
de libErable dEposEe sur une surface interne
de la cellule et situEe au contact dudit echantil-

25 Ion quand ledit Echantillon est introduit dans

ladite cellule et a I'intErieur de ladite zone
rEactionnelle, ledit rEactif redox Etant HbErE
dans la solution dans ledit Echantillon aqueux
dans ladite zone Etant mis h rEagir directement

30 ou indirectement avec ledit composant pour

former une substance oxydable ou rEductible
Electrochimiquement dans ladite zone, ladite
substance oxydable ou rEductible Etant capa-
ble de rEagir electrochimiquement avec ladite

35 Electrode et Etant diffErente dudit rEactif redox.

9. Appareil selon la revendication 8, comprenant
de plus une enzyme situEe au contact dudit
echantillon quand ledit echantillon est introduit

40 dans ladite cellule et a I'intErieur de ladite

zone rEactionnelle, ladite enzyme Etant capa-
ble de catalyser directement ou indirectement
la rEaction entre ledit composant et ledit reactif
redox.

45

10. Appareil selon la revendication 9, dans lequel
ladite enzyme est portEe com me une couche
libErable sur une surface de ladite cellule a
reaction ou zone rEactionnelle qui en utilisation

so est en contact avec TEchantillon aqueux intro-

duit dans ledit appareil.

11. Appareil selon la revendication 9 ou 10, dans
lequel ladite enzyme comprend une oxydorE-

55 ductase.

12. Appareil selon la revendication 11, dans lequel
ladite enzyme comprend la glucose oxydase.

8

13

EP 0 255 291 B1

14

13. Appareil selon Tune quelconque des revendi-
cations 8 a 12, dans lequel ledit reactif redox
est choisi parmi le groupe constitue* de ferri-
cyanure, du bleu de methylene, du benzoqui-
none, du 2,6-dichlorophe'nolindophgnol, de la 5
pyocyanine et de la thionine, et ladite substan-
ce oxydable ou reductible Slectrochimique- 4.
ment est oxydable et comprend une forme
rSduite dudit rSactif redox.

14. Appareil selon I'une quelconque des revendi-
cations 8 a 13, dans lequel lesdites electrodes
sont choisies parmi le groupe constitue* d'elec-
trodes en or, en carbone et en argent.

Patentansprtiche

1. Verfahren zum Messen oder Feststellen einer
Komponente einer waBrigen Flussigkeitsprobe,
das umfaBt:

das EinfOhren einer Probe, die die interes-
sierende Komponente moglicherweise enthalt,
in eine Reaktionszelle zur kapillaren Fullung,
wobei die Reaktionszelle mindestens zwei fur
coulometrische Messungen geeignete Elektro-
den umfaBt, so daB die Probe eine Fussigkeits-
schicht einer Dicke von weniger als etwa 0.2
mm in einer Reaktionzone bildet, die uber ei-
ner der Elektroden liegt:

das direkte oder indirekte Umsetzen dieser
gegebenenfalls vorliegenden Komponente mit
einem Redox-Reagenz, das anfanglich in Form
einer festen, freisetzbaren Schicht vorliegt, die
auf einer inneren Oberflache der Reaktionszel-
le abgeschieden ist und in die Losung in der
Probe in der Zone freigesetzt wird, wodurch in
dieser Zone in einer Menge, die der Menge
dieser in der Zone vorliegenden Komponente
Proportional ist, eine elektrochemisch oxidier-
bare oder reduzierbare Substanz gebildet wird,
die elektrochemisch mit der Elektrode reagie-
ren kann und von dem Redox-Reagenz ver-
schieden ist, und

das coulometrische Feststellen oder Be-
stimmen einer Menge an elektrischer Ladung,
die sich auf die oxidierbare oder reduzierbare
Substanz in dieser Zone bezieht, urn so ein
Anzeichen fur die Menge der interessierenden
Komponente zu ergeben.

2. Verfahren gemaB Anspruch 1, worin die zu
messende Komponente durch anfangliche en-
zymatische oder chemische Umwandlung ei-
nes Analyten gebildet wird.

3. Verfahren gemaB Anspruch 1 oder 2, worin
das Redox-Reagenz aus der aus Cyanoferrat-
(III), Methylenblau, p-Benzochinon, 2,6-Di-

chlorphenolindophenol, Pyocyanin und Thionin
bestehenden Gruppe ausgewahlt wird und die
elektrochemisch oxidierbare oder reduzierbare
Substanz oxidierbar ist und eine reduzierte
Form des Redox-Reagenzes umfaBt.

Verfahren gemaB irgendeinem der AnsprUche
1 bis 3, worin die Komponente mit einem En-
zym und dem Redox-Reagenz umgesetzt wird,
w um so die elektrochemisch oxidierbare oder

reduzierbare Substanz zu bilden.

5. Verfahren gemaB Anspruch 4, worin das En-
zym ein Oxidoreduktaseenzym umfaBt.

75

6. Verfahren gemaB Anspruch 4 oder 5, worin die
Komponente einen reduzierenden Zucker um-
fa/Jt und das Enzym eine entsprechende Zuck-
eroxidase umfaBt.

20

7. Verfahren gemaB Anspruch 6, worin die Kom-
ponente Glucose umfaBt und das Enzym Glu-
coseoxidase umfaBt.

25 8. Vorrichtung zur Durchfuhrung einer elektroche-
mischen Feststellung oder Messung einer
Komponente einer waBrigen Flussigkeitsprobe,
umfassend:

eine Reaktionszelle mit kapillarer Fullung,

30 die mindestens zwei Elektroden umfaBt, die fur

die coulometrische Messung geeignet und in
einer Reaktionszone Oder benachbart dazu an-
geordnet sind, wobei die Reaktionszone eine
waBrige Flussigkeitsprobe, die die interessie-

35 rende Komponente moglicherweise enthalt,

aufnehmen kann, und wobei die Elektoden und
die Reaktionszone so angeordnet sind, daB
FIQssigkeit in der Zone die Elektroden beruhrt
und eine Schicht einer Dicke von weniger als

40 etwa 0,2 mm bildet, die uber einer der Elektro-
den liegt;

und die Zelle auch ein Redox-Reagenz in
Form einer festen freisetzbaren Schicht um-
faBt, die auf einer inneren Oberflache der Zelle

45 abgeschieden ist und angeordnet ist, um mit

der Probe in BerUhrung zu kommen, wenn
diese in die Zelle und in die Reaktonszone
eingefuhrt wird, wobei das Redox-Reagenz in
die Losung in der waBrigen Probe in der Zone

so freigesetzt wird und mit der Komponente direkt

oder indirekt unter Bildung einer elektroche-
misch oxidierbaren oder reduzierbaren Sub-
stanz in der Zone reagiert, wobei die oxidierba-
re oder reduzierbare Substanz elektrochemisch

55 mit der Elektrode reagieren kann und von dem

Redox-Reagenz verschieden ist.

9. Vorrichtung gemaB Anspruch 8, die femer ein

9

15 EP 0 255 291 B1 16

Enzym umfaflt, das angeordnet ist, urn mit der
Probe in Kontakt zu kommen, wenn diese in
die Zelle und in die Reaktionszone eingefOhrt
wird, wobei das Enzym fahig ist, eine direkte
Oder indirekte Reaktion zwischen der Kompo- 5
nente und dem Redox-Reagenz zu katalysie-
ren.

10. Vorrichtung gemSB Anspruch 9, worin das En-
zym als eine freisetzbare Schicht auf einer w
Oberflache der Reaktionszelle Oder Reaktions-
zone vorhanden ist, die bei Verwendung von

der in die Vorrichtung eingefOhrten waflrigen
Probe beruhrt wird.

75

11. Vorrichtung gemafi Anspruch 9 oder 10, worin
das Enzym eine Oxidoreduktase umfaflt.

12. Vorrichtung gemafl Anspruch 11, worin das
Enzym Glucoseoxidase umfaflt. 20

13. Vorrichtung gemafl irgendeinem der AnsprU-
che 8 bis 12, worin das Redox-Reagenz aus
der aus Cyanoferrat(IM), Methylenblau, p-Ben-
zochinon, 2,6-Dichlorphenolindophenol, Pyocy- 25
anin und Thionin bestehenden Gruppe ausge-
wahlt ist und die elektrochemisch oxidierbare
oder reduzierbare Substanz oxidierbar ist und
eine reduzierte Form des Redox-Reagenzes
umfa/3t. 30

14. Vorrichtung gem§0 irgendeinem der AnsprO-
che 8 bis 13, worin die Elektroden aus der
Gold-, Kohienstoff- und Silberelektroden um-
fassenden Gruppe ausgewahlt sind. 35

40

50

55

10